a n a l y t i c a c h i m i c a a c t a 6 0 0 ( 2 0 0 7 ) 122–128
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/aca
On-line coupling of sequential injection extraction with
restricted-access materials and post-column derivatization
for sample clean-up and determination of propranolol
in human plasma
ˇ ınsky´ a,∗ , Hugo S. Serralheiro b , Petr Solich a ,
Dalibor Sat´
´ b , Maria C.B.S.M. Montenegro b
Alberto N. Araujo
a
Department of Analytical Chemistry, Faculty of Pharmacy, Charles University, Heyrovsk´eho 1203,
´ e 500 05, Czech Republic
Hradec Kralov´
b Requimte, Departamento de Qu´ımica-F´ısica, Faculdade de Farmacia,
´
Universidade do Porto,
R. An´ıbal Cunha 164, Porto 4070-047, Portugal
a r t i c l e
i n f o
a b s t r a c t
Article history:
The presented paper deals with a new methodology for direct determination of propranolol
Received 16 October 2006
in human plasma. The methodology described is based on sequential injection analy-
Received in revised form
sis technique (SIA) coupled with solid phase extraction (SPE) column based on restricted
6 February 2007
access materials (RAM). Special RAM column containing 30 ␮m polymeric material—N-
Accepted 10 February 2007
vinylacetamide copolymer was integrated into the sequential injection manifold. SIA–RAM
Published on line 20 February 2007
system was used for selective retention of propranolol, while the plasma matrix components
were eluted with two weak organic solutions to waste.
Keywords:
Due to the acid–basic and polarity properties of propranolol molecule and princi-
Sequential injection analysis
ples of reversed-phase chromatography, it was possible to retain propranolol on the
Solid phase extraction
N-vinylacetamide copolymer sorbent (Shodex MSpak PK-2A 30 ␮m (2 mm × 10 mm)).
Restricted access materials
Centrifuged plasma samples were aspirated into the system and loaded onto the column
Propranolol
using acetonitrile–water (5:95, v/v), pH 11.00, adjusted by triethylamine. The analyte
Human plasma
was retained on the column while proteins contained in the sample were removed to
Sample preparation
waste. Interfering endogenous substances complicating detection were washed out by
acetonitrile–water (15:85), pH 11.00 in the next step. The extracted analyte was eluted by
means of tetrahydrofuran–water (25:75), pH 11.00 to the ﬂuorescence detector (emission
ﬁlter 385 nm). The whole procedure comprising sample pre-treatment, analyte detection
and column reconditioning took about 15 min. The recoveries of propranolol from undiluted
plasma were in the range 96.2–97.8% for three concentration levels of analyte. The proposed
SIA–RAM method has been applied for direct determination of propranolol in human
plasma.
© 2007 Elsevier B.V. All rights reserved.
∗
Corresponding author. Tel.: +420 495067228; fax: +420 495067164.
ˇ ınsky).
´
E-mail address: [email protected] (D. Sat´
0003-2670/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2007.02.021
a n a l y t i c a c h i m i c a a c t a 6 0 0 ( 2 0 0 7 ) 122–128
1.
123
Introduction
Sequential injection analysis (SIA) was introduced by Ruzicka and Marshall in 1990 [1] as a new generation in the
development of ﬂow injection technique. The principles upon
which SIA is based, namely controlled partial dispersion
and reproducible sample handling, are similar to those of
the ﬂow injection analysis (FIA). Characteristic advantages
of SIA include its versatility, full computer compatibility, high sample throughput, and low sample and reagent
consumption.
However, in recent years, it has become apparent that
the scope of SIA can be extended to encompass a variety
of more complex, on-line sample-manipulation and pretreatment procedures. Then, the ports of the multi-position
selection valve could be coupled to various units (e.g., reservoirs, detectors, pumps, reactors, separators, special cells, and
other manifolds) [2]. One of the most widely used samplepre-treatment procedures in SIA is the automated solid-phase
extraction (SPE). It employs an appropriate solid- or liquidextraction material attached to a suitable support, for which
arrangement SIA is ideally suited. The extraction procedure
accomplishes two goals: separation of the analyte from interfering species in the sample and preconcentration of the
analyte to increase the sensitivity. The great advantage of SPE
is that both organic compounds and inorganic species can be
extracted. Depending on the nature of analyte and on required
retention mechanism, different extraction materials can be
used (e.g., hydrophilic ion-exchange resins, surface-modiﬁed
beads, molecularly imprinted polymers and different types of
hydrophobic polymers) [2].
In recent years, special SPE supports possessing restricted
access properties have been developed to allow direct injection of untreated biological samples into on-line SPE liquid
chromatography (LC) systems [3–5]. These sorbents called
restricted access materials (RAMs), combine size exclusion
of proteins (without destructive accumulation) and other
macromolecular matrix components with the simultaneous
enrichment of low-molecular analytes, which can be retained
and extracted selectively.
The low-molecular-mass analytes are retained by conventional retention mechanisms such as hydrophobic, ionic
or afﬁnity interactions at the inner surface of the sorbent
particles. The access of proteins is prevented by a physical
diffusion barrier or by a chemical barrier. In the majority
of applications described in the literature, RAMs are used
in on-line coupled-column SPE–LC systems. These systems
require highly sophisticated apparatus, higher operating cost
and expensive instrumentation (column switching systems,
software control and two pumps). Coupling of SPE based on
restricted access materials with SIA was described as low cost
alternative for direct determination of drugs. First attempt to
on-line coupling of RAM and SIA was performed by determination of potential antileucotrienic quinlucast in serum and
it provided satisfactory results [6] that encouraged following
studies of the hyphenation. The aim of presented work was
to examine further possibilities of SIA–RAM system for direct
analysis of propranolol in human plasma with ﬂuorescence
detection.
Fig. 1 – Molecular structure of propranolol.
Propranolol (structure shown in Fig. 1) was chosen as an
analyte possessing ﬂuorescence capability and representing
polar and basic substances from the group of beta-blocking
pharmaceuticals. Being a strong beta-adrenergic blocking
drug, propranolol is widely used in clinical practice in the
treatment of cardiac arrhythmia, hypertension, sinus tachycardia and angina pectoris [7]. It is also used in low activity
sports, reducing cardiac frequency, contraction force and coronary ﬂow [8]. Therefore, it has been included in the list of
forbidden substances by International Olympic Committee [9].
Monitoring of propranolol in bio-ﬂuids is important not only
in the clinical practice but also in the ﬁeld of doping control.
Different techniques [10–16], including ﬂuorimetry, HPLC,
capillary electrophoresis and mass spectrometry, have been
used to determine propranolol in commercial formulations or
biological ﬂuids.
Practically, all previous methods operate in a ﬂow or batch
systems and require various tedious preliminary procedures
such as pre-concentration in an organic solvent. Thus, in
recent years new techniques to determine propranolol such
as ﬂuorescence optosensors [17], molecularly imprinted polymers [18] or ion selective PVC membrane electrodes [19]
have been developed, but it is still necessary to further
develop highly selective, simple, rapid and cheap procedures
to determine propranolol in pharmaceutical preparations and
bio-ﬂuids.
Method for direct determination of propranolol in human
plasma comprising on-line sample preparation based on
SIA–RAM hybrid technique has been proposed. Advantages
and disadvantages of such a connection are reported in presented paper.
2.
Experimental
2.1.
Sequential injection system and RAM sorbents
A commercially available instrument FIAlab® 3500 system
(FIAlab® Instruments Inc., Bellevue, USA) with a syringe pump
(syringe reservoir 5.0 mL) and an 8-port selection Cheminert
valve (Valco Instrument Co., Houston, USA) was used. The
manifold was equipped with ﬁbre-optic ﬂuorimetric detector
PMT-FL (Ocean Optics Inc., Dunedin, USA) with UV light source
D-1000-CE (Analytical Instrument Systems Inc., Flemington,
USA). The ﬂuorescence signal was scanned through secondary
ﬁlter (385 nm, Edmund Industrie Optik, GmbH, Karlsruhe, Ger-
124
a n a l y t i c a c h i m i c a a c t a 6 0 0 ( 2 0 0 7 ) 122–128
Fig. 2 – Scheme of SIA–RAM system with post-column derivatization.
many). The SIA system was equipped with ﬂow rate variable
peristaltic pump. The whole SIA system was controlled by
the version of program FIAlab for Windows 98, WinFIA version 5.0. Flow lines were made of 0.75 mm i.d. PTFE tubing.
On-line sample preparation was performed on RAM column
˚ pore size (Shodex,
MS Pak PK-2A (30 ␮m, 2 mm × 10 mm), 30 A
Japan). The RAM column was placed between the selection
valve and ﬂow cell of the detector. A replaceable in-line ﬁlter
(2–5 ␮m, Merck) was installed ahead the column for protection.
The next sorbent used in our study was LiChrospher® RP18 ADS (alkyl-diol silica) (25 ␮m, 25 mm × 4 mm), from Merck
(Germany). A scheme of the sequential injection extraction
system with the RAM column and post-column derivatization
is depicted in Fig. 2.
2.2.
Reagents
All chemicals used were of analytical grade quality. Propranolol and organic solvents were obtained from Sigma–Aldrich.
Chemicals for buffer preparation were obtained from Merck,
Germany. Millipore Milli-Q RG (Millipore s.r.o., Prague, Czech
Republic) ultra pure water was used for preparing the solutions. Eluting solutions were degassed by helium before use.
2.3.
Preparation of spiked human plasma and
standard samples
A stock solution of propranolol (1000 ␮g mL−1 ) was prepared
by dissolving the substance in methanol. The ﬂask was stored
in the refrigerator protected to light for 2 weeks without
stability problems. Fresh working standard solutions were prepared daily by appropriate dilution of the stock solution in
water to the concentration 20 ␮g mL−1 . The sample of human
plasma was spiked with stock solution of propranolol to get
the ﬁnal concentrations of propranolol (1, 5, 10 ␮g mL−1 ) in
undiluted plasma. The samples were spiked just before analysis, incubated for 1 h at 37 ◦ C and then centrifuged for 10 min
at 1750 × g. The supernatant was used for the analysis.
2.4.
Design of proposed SIA–RAM analytical procedure
A procedure based on sequential aspiration of mobile phases
of increasing content of organic modiﬁer and their pro-
pelling through the column was proposed. The analytical
cycle involved four main steps: (1) loading the sample onto
the column and removing proteinaceous ballast material; (2)
washing the column and removing more polar interfering substances complicating detection; (3) elution and detection of
the analyte; and (4) column reconditioning. The composition
of mobile phases used for particular steps was optimised separately.
First, the syringe pump was ﬁlled with loading mobile
phase acetonitrile–water (5:95, v/v), pH 11.00 (adjusted by triethylamine) via the left position of the double position valve
A. The sample (standard solution or spiked plasma solution,
50 ␮L) was aspirated via port 5 of the selection valve B (switching the valve A to the right position) to the connecting tube
leading from the middle port of the valve B to the pump.
The sample was then propelled through port 8 of the selection valve B to the RAM column by reverse movement of the
piston pump using ﬂow rate 0.6 mL min−1 . Propranolol was
extracted on the column while proteinaceous matrix of the
sample was washed to waste. In the next step, washing mobile
phase acetonitrile–water (15:85), pH 11.00 (adjusted by triethylamine) was aspirated via port 4 of the selection valve B
and pushed through the column washing ﬂuorescent interfering substances to waste (ﬂow rate 1.2 mL min−1 ). Finally,
eluting mobile phase tetrahydrofuran–water (25:75), pH 11.00
(adjusted by triethylamine) was aspirated via port 2 of the
selection valve B and it was used for elution of extracted
propranolol to the mixing coil for post column derivatization before ﬂuorimetric detection (ﬂow rate 1.2 mL min−1 ). The
aspiration of derivatization reagent was carried out by peristaltic pump-ﬂow rate 0.045 mL min−1 . It was necessary to
wash the column with 80% acetonitrile (0.5 mL) and recondition it with the loading mobile phase (0.6 mL) (both ﬂow rates
1.2 mL min−1 ) prior to aspiration of the next proteinaceous
sample. The latter two steps were integrated at the beginning
of the controlling program to ensure that the column will be
prepared for the following cycle.
The resulting signal was recorded in the form of peaks;
the peak heights were calculated automatically by FIAlab®
software and the data were stared by PC for subsequent processing. Each measuring cycle was carried out in triplicate and
the mean peak height values were used for data evaluation. All
measurements were performed at ambient temperature.
a n a l y t i c a c h i m i c a a c t a 6 0 0 ( 2 0 0 7 ) 122–128
3.
Results and discussion
3.1.
Choice of the extraction column
The experiments concerning a composition of mobile phases
started searching the optimal solvent for ﬁnal elution
of propranolol. First experiments were carried out with
LiChrospher® RP 18 ADS column. The optimum composition of
eluting mobile phase was not found for this type of sorbent.
The main problem of retention versus extraction of propranolol from LiChrospher® RP 18 ADS was in molecular structure
of the drug. Propranolol shows relatively high lipophilicity
coefﬁcient (log P = 2.60) [20], which is suitable for sufﬁcient
retention on reversed phase C-18 sorbent. However, basic character of the propranolol molecule (pKa = 9.15) [20] resulted in
a poor retention in working pH area of LiChrospher® RP 18
ADS column (pH 2–7). Moreover, the elution of propranolol
from silica based RP 18 sorbent showed very strong peak
tailing. The retention behaviour of propranolol was checked
making different changes in the composition of the eluting
mobile phase and observing the changes in retention time
and shape of the peak. The tested organic modiﬁers were
methanol, ethanol, acetonitril, propanol or tetrahydrofuran
in different ratios with buffers in pH range from 2 to 7. The
way to improve peak tailing on LiChrospher® RP 18 ADS sorbent was not found under the tested conditions. Peak shape
of propranolol obtained on this sorbent could not be used
for analytical evaluation. The value of peak asymmetry factor of propranolol was higher than 4.0. The main task of the
following experiments was to obtain a symmetric peak of propranolol free of the noise of dead volume of the system. The
next tested extraction column was polymeric material—Nvinylacetamide copolymer sorbent (Shodex MSpak PK-2A
(30 ␮m, 2 mm × 10 mm)). Compared to LiChrospher® RP 18
ADS column, Shodex MSpak PK-2A sorbent can be used in
a wide pH range (pH 2–12). This sorbent was found suitable
for the extraction of propranolol from human plasma. Extraction process was carried out at pH 11 regarding the acid–base
properties of propranolol molecule. Symmetric peak of propranolol free of the noise of dead volume of the system was
obtained during the elution step with optimal mobile phase
tetrahydrofuran–water (25:75), pH 11.00 (adjusted by triethylamine).
3.2.
Optimization of the steps of the sequential
injection extraction
A procedure based on sequential aspiration of different washing and eluting mobile phases and their propelling through the
column was proposed. The priority of the study was to optimise the composition of all mobile phases to obtain a peak
of the analyte that was differentiated both from the matrix of
serum and from the disturbing peak of dead volume of mobile
phase after the change of phases in the ﬂow cell. The mobile
phases used in particular steps inﬂuenced retention of the
analyte and matrix during the whole procedure. To optimize
their composition required a complex process. The optimization of the single steps discussed in the following text was
based on ﬁnding the optimum composition of eluting mobile
125
phase and adapting the composition of other mobile phases
to these conditions.
Loading mobile phase is proposed for propelling of the
sample to the column and subsequent elution of proteins
from column. Content of organic phase is limited because
of the risk of proteins denaturation and clogging the system. However, addition of low amount of organic solvent is
recommended to effectively disrupt the drug and serum proteins interactions and to enhance the selectivity and sample
clean up. Methanol–water (2:98, v/v) was tested but using this
loading mobile phase for protein matrix elution resulted in
slow and poor elution of proteinaceous ballast matrix. Finally,
acetonitrile–water (5:95, v/v), pH 11.00, adjusted by triethylamine was employed as loading mobile phase with good
results concerning the recovery of the analyte.
Washing step using washing mobile phase had to be
inserted between loading of the sample onto the column
and elution of analyte to the detector in order to remove
some interfering ﬂuorescent substances to waste prior to
analyte detection and quantitation. A compromise between
sufﬁcient strength of washing solution for removing the interfering components and low impact of this solution on the
retention of propranolol during elution step had to be found.
Acetonitrile–water (15:85, v/v) pH 11.00, adjusted by triethylamine was ascertained to be the optimal washing mobile
phase that did not negatively inﬂuence retention of propranolol. Washing mobile phase of this composition enabled
complete removing of ﬂuorescent interferences from undiluted plasma. Complete sample clean-up procedure was
achieved before elution of propranolol to detector.
It was necessary to adjust the composition of eluting
mobile phase to obtain convenient conditions for retention
of propranolol at the column and its elution in form of a
symmetric peak separated from the peak of dead volume
after changing mobile phases in the ﬂow cell. Due to the
limited length of the Shodex MSpak column (10 mm) and
high particles size (30 ␮m) there was a tendency to peak
broadening and tailing with lower content of organics in
mobile phase. On the other hand, increase of the amount
of organics in mobile phase led to co-elution of propranolol
with the dead volume. Various types of organic modiﬁers
(methanol, acetonitrile, isopropanol, tetrahydrofuran) in different ratios in water were tested. Triethylamine was added to
the mobile phase in attempt to suppress the ionization of propranolol during the extraction process. Finally mobile phase
tetrahydrofuran-water (25:75, v/v), pH 11.00 (adjusted by triethylamine) provided the best conditions for elution of analyte
with respect to the retention time of propranolol, peak width
and peak symmetry.
Acetonitrile–water (80:20, v/v) was used for column cleanup and conditioning between the single analytical cycles. It
was necessary to recondition the column with loading mobile
phase prior to injection of the next sample to prevent denaturation of the plasma proteins in column.
3.3.
Flow rates and volumes of the mobile phases in
the particular steps of the procedure
SIA is generally characterised by short time of analysis
and high sample throughput. However, in case of SIA–RAM
126
a n a l y t i c a c h i m i c a a c t a 6 0 0 ( 2 0 0 7 ) 122–128
extraction the whole analytical procedure is more complex
comprising sample pre-treatment and analyte quantitation
and thus sample throughput is substantially reduced. Flow
rates used with this technique are limited by back pressure of
the RAM column, especially by using longer columns or lower
particle size.
Flow rate of 0.6 mL min−1 was proposed for the step of
sample loading and for removing the macromolecular ballast
to waste. Using this ﬂow rate prevented overloading of the
syringe pump and provided enough time for interactions of
the analyte with sorbent. Volume of the loading mobile phase
is a crucial parameter. To remove all proteinaceous matrix
from the column prior to aspiration of washing mobile phase
with higher content of organic phase (denaturating properties)
is necessary. Contamination of the column with precipitated
proteins would lead to an irreversible increase in back pressure
and decrease in capacity and selectivity of the column. Removing of proteinaceous ballast was monitored using ﬂuorimetric
detector. The ﬁrst step of the procedure (loading of sample
and removing proteins from the column) was considered to be
ﬁnished when the detector had reached the baseline. A total
volume of 4.9 mL of loading mobile phase corresponding to
time 490 s after injection was found to be sufﬁcient for complete removing of proteinaceous matrix after injection of 50 ␮L
of undiluted plasma. The ﬂuorescence signal after injection of
50 ␮L of undiluted plasma did not reach the baseline within
time ascertained for fractionation step, which indicates presence of some low-molecular interfering substances that have
to be removed in the next step.
The second washing mobile phase −4.0 mL of acetonitrile–
water (15:85, v/v) pH 11.00 enabled complete removal of interfering substances without inﬂuencing the retention of the
analyte. Flow rate was increased to 1.2 mL min−1 for this washing step, which was a compromise between the speed of
analysis and washing efﬁciency.
Elution mobile phase tetrahydrofuran–water (25:75, v/v),
pH 11.00 was propelled through the column at ﬂow rate of
1.4 mL min−1 without any problems concerning the back pressure. A higher value of ﬂow rate also resulted in a narrower
and higher peak of analyte comparing to lower ﬂow rates. The
lowest volume ensuring the complete elution of the analyte
(5.0 mL) was applied. Flow rate 1.2 mL min−1 was used for column clean-up and conditioning between the single analytical
cycles.
3.4.
pH conditions and ﬂuorescence
Due to the acid–base properties of propranolol (pKa 9.15), it was
necessary to increase pH of eluting mobile phase up to values
at which ionisation of propranolol is suppressed and analyte is
well adsorbed to N-vinylacetamide copolymer of the sorbent.
Sufﬁcient extraction of propranolol was achieved with eluting
mobile phase at pH 11.00. High pH was crucial for shape of
peak of the eluted analyte. pH of the loading and the washing
mobile phase was adjusted to 11.00 for the same purpose.
The effect of pH value on the ﬂuorescence intensity was
tested without RAM column in the wide range pH values
2–12. The solutions tested were 0.01 M HCl; phosphate buffers
(0.05 M KH2 PO4 and 0.05 M K2 HPO4 ) and 0.01 M NaOH. These
solutions were mixed with the zone of propranolol directly
Fig. 3 – Effect of the ﬂow rate of peristaltic pump and HCl
concentration on ﬂuorescence intensity of propranolol.
in the system and they were transferred through the ﬂuorescence detector. Signiﬁcant increase in ﬂuorescence signal of
propranolol was observed in the acid area. Changing pH to
alkaline area (pH 6 and higher) resulted in strong decrease in
ﬂuorescence signal about 90%.
The solution of the mentioned problem was found in post
column derivatization of propranolol. Main task of this procedure was to decrease pH of the elution zone of propranolol
behind the column. T-point connection and mixing coil (15 cm
length) where ﬂow from the column was mixed with an acid
solution from a peristaltic pump was used. Different concentrations of HCl solutions (0.001 M, 0.01 M and 0.1 M) were
tested and 0.1 M HCl was found to be optimal. The ﬂow rate of
peristaltic pump was adjusted to 0.045 mL min−1 . There was
necessary to ﬁnd a compromise between the concentration of
HCl and ﬂow rate of the peristaltic pump. Higher ﬂow rates of
peristaltic pump resulted in dilution of sample zone. Therefore, the use of higher concentration of HCl with combination
of lower ﬂow rate of peristaltic pump showed a better sensitivity of the determination. The results of ﬂow rates and HCl
concentrations optimization are depicted in Fig. 3.
3.5.
Validation
The method was validated with respect to linearity, precision,
accuracy, selectivity and sensitivity in order to evaluate the
reliability of results provided by method.
3.5.1.
Calibration and sensitivity
The calibration curve was established by measuring the ﬂuorescence signal of seven solutions of various concentrations
of propranolol in plasma. The linear relation between ﬂuorescence intensity and concentration of propranolol was found in
the range 1.0–75 ␮g mL−1 and was described by following equation: IF = (7506 ± 33)c + (80 632 ± 1183); (IF means intensity of
ﬂuorescence, c concentration of propranolol), the correlation
coefﬁcient being 0.9999.
The limit of detection and limit of quantitation were calculated by comparison of three-fold (3) and 10-fold (10)
a n a l y t i c a c h i m i c a a c t a 6 0 0 ( 2 0 0 7 ) 122–128
127
sible to carry out a sample throughput of four samples per
hour.
4.
Fig. 4 – Record of blank human plasma (A) and peak of
propranolol (5 ␮g mL−1 ) in undiluted plasma (B); 50 ␮L of
sample injection, tetrahydrofuran–water (25:75, v/v), pH
11.00, ﬂow rate 1.4 mL min−1 , ﬂuorescence detection.
variation, respectively, of baseline noise and signals of plasma
samples spiked with known concentrations of propranolol.
The detection limit was 0.046 ␮g mL−1 ; the limit of quantitation was estimated to be 0.15 ␮g mL−1 for 50 ␮L of undiluted
plasma injection. The sensitivity of system was sufﬁcient for
real drug level monitoring of propranolol in human plasma of
patients.
3.5.2.
Precision
Precision of proposed procedure was characterised by parameter of repeatability, which was calculated for eight consecutive
measurements at three concentration levels 1, 5 and
10 ␮g mL−1 of propranolol in spiked plasma samples. Relative standard deviations (R.S.D.) were 0.54%, 2.1% and 0.6%,
respectively. The inter-day repeatability was determined at
one concentration level 5 ␮g mL−1 of propranolol standard
water solution for three consecutive days. The result in form
of R.S.D. was determined less than 4.0% (n = 8).
3.5.3.
Accuracy
Accuracy of the method was expressed as a parameter of
recovery. The recovery was calculated at three concentration
levels (1, 5 and 10 ␮g mL−1 ) by comparison of responses of propranolol from spiked plasma samples with those found by
injection of standard solutions at the same concentrations.
The mean absolute recovery lay between 96.2 and 97.8% for
propranolol and extraction efﬁciency was relatively constant
over the range mentioned above (R.S.D. less than 5.2%). RAM
column could be used repeatedly in the SIA system without
loss of extraction efﬁciency.
A hybrid SIA–RAM technique with post column derivatization
and ﬂuorescence detection of propranolol has been proposed.
The RAM column can be used repeatedly and enables one to
remove interfering substances and to determine propranolol
directly in human plasma. The proposed SIA–RAM method
involving sample preparation can be simply automated and
offers the possibility of restriction of manual sample handling. Minimum manipulation with the biological sample
results in improved precision and accuracy, shorter analysis time and lower costs per analysis. In contrast to HPLC
with off-line SPE technique, SIA–RAM technique is based on
non-continuous ﬂow, which enables reduction of volumes of
consumed reagents and produced waste. The method exhibits
similar sensitivity as conventional separation techniques and
requires less expensive instrumentation. SIA–RAM may well
be a good alternative to more sophisticated techniques for
the analysis of biological samples, controlling concentrations
of pharmaceutical preparations and for screening of patient
compliance or doping control. In the case of analysis of biological material it mostly enables to determine only the total
amount of the original analyte and its metabolites, not the
precise amounts of particular substances.
Acknowledgments
The authors gratefully acknowledge the ﬁnancial support
of the Grant Agency of Czech Republic, project no. GA CR
203/05/P164 and Czech Ministry of Education project MSM
0021620822.
references
[1]
[2]
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3.5.4.
Selectivity
The absence of interfering endogenous components of plasma
during elution of propranolol is demonstrated in Fig. 4 (A).
Propranolol contained in the plasma sample was adsorbed
to the column while the interfering ﬂuorescence substances
were removed to waste. Analyte was then eluted from column separately and provided ﬂuorescence signal, which could
be used for determination of propranolol. Fig. 4 shows typical record obtained during elution step of blank plasma,
and elution step by analysis of plasma spiked with propranolol (5 ␮g mL−1 ). Washing of interfering components
from column was achieved with two mobile phases, ﬂow
rates 0.6 mL min−1 and 1.2 mL min−1 , therefore it was pos-
Conclusion
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